Methods for delaying leaf senescence using the ORE7 gene

ABSTRACT

The present invention relates to a gene regulating leaf longevity of plants and a method for regulating the longevity of plants using the same. More particularly, it relates to a ORE7 gene regulating leaf longevity of plants which has a nucleotide sequence represented by SEQ ID NO: 1, and to a method for regulating the longevity of plants, in which the ORE7 gene is introduced into the plants and overexpressed, thereby delaying senescence of the plants. Plants can be transformed with ORE7 gene according to the present invention, so that the longevity of the plants is extended, thereby improving productivity and storage efficiency of the plants. Furthermore, the ORE7 gene and an ORE7 protein expressed therefrom according to the present invention are useful for studies of senescence mechanisms, and for identification of senescence-associated genes or senescence inhibitory substances, in plants.

TECHNICAL FIELD

The present invention relates in general to a gene regulating leaflongevity of plants and a method for regulating the longevity of plantsusing the same. More particularly, it relates to a gene ORE7 regulatingleaf longevity of plants which has a nucleotide sequence represented bySEQ ID NO: 1, and to a method for regulating the longevity of plants, inwhich the gene ORE7 is introduced into the plants and overexpressed,thereby delaying senescence of the plants.

BACKGROUND ART

Senescence is the final stage that plants undergo during their lifetime.The initiation of senescence can be said to be a rapid changeover pointin a plant's development stage. As senescence progresses, a plant'ssynthesis ability gradually decreases and it loses cellular homeostasiswith successive degradation of intracellular structures andmacromolecules, finally leading to death (Matile P. et al., Elservier,413–440, 1992; Nooden L. D. et al., Academic press, 1988; Thiman K. V.et al., CRC press, 85–115, 1980; and Thomas H. et al., Annu. Rev. PlantPhysiol., 123:193–219, 1993). Such senescence of plants is a series ofcontinuous biochemical and physiological phenomena, which is geneticallydestined to progress in highly intricate and active manners at cell,tissue and organ levels. However, the senescence of plants is seen as aprocess of cellular degeneration, and at the same time, a geneticcharacter which is actively acquired for adaptation to environmentduring the development process, including migration of nutrients fromgrowth organs to genital organs at the winter season.

The suppression of plant senescence is not only of great scientificimportance in itself, but also of great industrial importance in termsof the productivity of crops or the possibility of improvingpost-harvest storage efficiency. For this reason, genetic, molecularbiological, physiological and biochemical studies have been activelyconducted in the attempt to establish plant senescence phenomena.However, reports regarding phytohormones are the main area of interest;studies on senescence regulation, such as the induction of senescenceregulation using senescence regulatory genes, are, as yet, insufficient.

Cytokinin, a plant growth hormone, is known as a hormone capable ofphysiologically delaying senescence. For this reason, there have beenstudies conducted to delay senescence by regulation of cytokininsynthesis, but there were problems in that other physiological actionsare also affected due to the influence of hormones. However, there hasbeen recent success in delaying the progression of senescence by amethod in which an IPT gene is linked to a promoter of asenescence-specific SAG12 gene so that the synthesis of cytokinin isspecifically regulated at a certain senescence stage. In the case oftobacco plants whose senescence was delayed by this method, an increaseof more than 50% in productivity could be achieved while causing littleor no changes in the blooming time and no other deformations (Gan S. etal., Science, 22:1986–1988, 1995). Moreover, plants for delay ofsenescence have been developed, making the ripe tomatoes a main objectof this development. For such development, the following methods havebeen applied; inhibition of synthesis of ethylene, a phytohormoneplaying an important role in senescence, or reduction of the amount ofintracellular ethylene (Klee et al., Plant Cell, 3(11): 1187–93, 1991;and Picton et al., Plant Physiol., 103(4): 1471–1472, 1993).

In addition, studies on delay of senescence are mainly focused on themanipulation of degradation-associated genes, which have activitiesassociated with biochemical changes occurring in a process of senescenceor are involved in the signal transduction system. A typical exampleconnected with such studies includes commercialized tomatoes, called“Flavr savr”, in which the expression of polygalacturonase gene involvedin the degradation of cell walls is impeded using antisense DNA so thatthe softening of tomatoes is prevented, thereby improving the transportand storage properties of tomatoes (Giovannoni et al., Plant Cell, 1(1):53–63, 1989). It was also reported that, where the expression ofphospholipase D involved in degradation of lipids is impeded with theantisense DNA, senescence caused by phytohormones is delayed (Fan etal., Plant Cell, 9(12): 2183–96, 1997). Furthermore, it was recentlyreported that leaf senescence is delayed, in tobacco plants in whichSAG12 promoter, and kn1 (knotted 1), a homeobox gene of corn, areexpressed (Ori et al., Plant Cell, 11:917–927, 1999).

However, a method capable of more directly regulating senescenceinvolves isolating mutant of senescence-associated genes and analyzinggenes which cause the mutation. According to existing reports, it isknown that, in Arabidopsis thaliana, the expression of ethylenereceptors is controlled in a ripening period of fruits or in thesenescence process of flowers (Payton S. et al., Plant Mol. Biol.,31(6): 1227–1231, 1996), and the expression of clp gene is regulated ina senescence process of leaves. Recently reported were studies on theidentification of genes involved in a senescence process of leaves (OhS. A. et al., Plant Mol. Biol., 30(4): 739–754, 1996), and on theisolation of leaf senescence-delaying mutants from Arabidopsis thaliana(Oh S. A. et al., The Plant Journal, 12(3):527–535, 1997). Also, amutant gene was successfully isolated from an ore9 mutant of the leafsenescence-delaying mutants (Woo H. R. et al., Plant Cell, 13:1779–1790, 2001). In addition, activities of a promoter of sen1, asenescence-associated gene, were reported (Oh S. A., et al., Journal ofPlant Physiology 151:339–345, 1997). However, studies on genes thatdirectly regulate senescence and their functions are, as yet,insufficient.

DISCLOSURE OF THE INVENTION

Therefore, the object of the present invention is to provide a generegulating leaf longevity of plants.

Another object of the present invention is to provide a method forregulating leaf longevity of plants using the gene.

To accomplish the object of the present invention, the present inventionprovides a gene ORE7 regulating the leaf longevity of plants, which hasa nucleotide sequence represented by SEQ ID NO: 1.

In addition, to accomplish another object of the present invention, thepresent invention provides a method for regulating the longevity ofplants, in which the gene ORE7 is introduced into the plants andoverexpressed, thereby delaying senescence of the plants.

As used herein, the term ‘longevity-extended mutant ore7’ or ‘ore7mutant’ means a mutant whose ORE7 gene is activated by an activationtagging method so that the mutant exhibits a senescence-delayingphenotype.

Hereinafter, the present invention will be described in detail.

In the present invention, in order to investigate genes involved inregulation of longevity in plants, mutants exhibiting thelongevity-extended character were selected. For this, Arabidopsisthaliana, frequently used as the subject for genetic and molecularstudies of plants, was used as a test plant.

Mutation induction methods which have generally been used to identifyfunctions of genes are specifically divided into the following threemethods: (1) A chemical method of inducing mutations by treatment withchemicals, such as ethyl-methyl sulfonic acid (EMS), (2) A method ofinducing mutation by X- or

-ray irradiation, and (3) A method of inducing mutation bytransformation with T-DNA of Agrobacterium, a soil bacterium. The methodof inducing the mutation by the chemical treatment, the X- or

-ray irradiation or the T-DNA insertion as described above, destroys apart or the whole of an original gene so that intrinsic functions of thegene are lost. Therefore, the functions of the genes can be deductedthrough the above methods. However, mutations formed by these methodsare mostly recessive mutations, and if other genes with functionssimilar to the destroyed gene are present in a genome, the phenotype isnot exhibited due to the other similar genes. Furthermore, if thedestroyed gene is a key, it is disadvantageous in that lethality ofplants may be caused.

On the contrary, the activation tagging method used in the presentinvention is advantageous in that it can induce the phenotype even whengenes with duplicate functions are present at other sites in genome ofArabidopsis thaliana, because T-DNA artificially activates theexpression of genes in plants without destroying a nucleotide sequenceof the genes (Weigel et al., Plant Physiology, 122:1003–1014, 2000).More specifically, the activation tagging method utilizes an activationtagging vector which has a selection marker, a replication originrequired for replication in E. coli, and an ampicillin-resistant gene,within T-DNA, in order to easily separate the surrounding DNA forming aboundary with T-DNA. Furthermore, the activation tagging vector has four35S CaMV enhancers in the right border of T-DNA. Therefore, when T-DNAis inserted into a genome of plants, it activates genes around sitesinserted with the T-DNA, thereby inducing mutations (see FIG. 6). Inthis case, even if other genes with similar functions are present ingenome, the phenotype can be exhibited by the genes activated by theenhancers. Therefore, when the activation tagging method is applied inorder to identify senescence regulatory genes, there is a highpossibility for investigation of novel senescence regulatory genes thatcannot be investigated by the loss-of-function mutation approach. Inaddition, the activation tagging method is advantageous in that itinduces dominant mutations so that the mutant phenotype is observed onegeneration in advance. Accordingly, the present inventors have selectedmutants with extended leaf longevity in Arabidopsis thaliana mutantsproduced by the activation tagging method, and investigated genesinvolved in an extended longevity of the selected mutants.

In one embodiment of the present invention, in order to select mutantswith extended leaf longevity in Arabidopsis thaliana, mutants wereproduced using an activation tagging vector pSKI015. Then, of the grownindividuals, an individual with a slow yellowing rate was selected withthe naked eye, and termed ‘ore7’. Following this, to verifylongevity-extending character of the ore7 mutant, it was examined forchanges in photosynthesis activity and chlorophyll content. Resultsindicated that, in the wild type, the photosynthesis activity and thechlorophyll content were completely lost at 40 days after germination(DAG), but in the longevity-extended mutant ore7, about 100% of thephotosynthesis activity and about 78% of the chlorophyll content weremaintained at the same period (see FIGS. 1B and 1C).

In another embodiment of the present invention, in order to verify thatan extension of longevity in the ore7 mutant is affected not only at aphysiological level but also at a molecular level, the expressionpatterns of various senescence-associated genes were examined bynorthern blot analysis. Results indicated that, in the wild type, theexpression of SEN4 and SAG12 genes, that are already known to increaseat a senescence stage, were increased rapidly as senescence progresses,whereas in the ore7 mutant, the expression of thesesenescence-associated genes were not substantially increased. On thecontrary, in the case of the wild type, the expression of chlorophylla/b binding protein gene which had been increased during anabolicaction, such as photosynthesis, was significantly decreased inproportion to the progression of senescence, but in the ore7 mutant ofthe present invention, there was little or no change in the expressionof this gene (see FIG. 1D).

Meanwhile, although leaf senescence is seen to be destined in genes, theprogression of senescence can be accelerated by dark treatment (Oh etal., Plant J., 12: 527–535, 1997). Therefore, the present inventorsexamined a change in leaf longevity of the ore7 mutant according to thedark treatment by measuring changes in photosynthesis activity andchlorophyll content. Examination of the progression of senescence causedby the dark treatment indicated that, in the wild type, the chlorophyllcontent and the photosynthesis activity were significantly decreased atabout 5 days after the dark treatment, but in the ore7 mutant, they weredecreased by only 3% and 22%, respectively (see FIGS. 2B and 2C).Furthermore, results of analysis for the expression of SEN4 gene, ameasure of senescence molecules, indicated that the expression of SEN4in the wild type was highly increased compared to the ore7 mutant (seeFIG. 2D).

Moreover, leaf senescence is known as being accelerated byphytohormones, such as abscisic acid (ABA), methyl jasmonate (MeJA) andethylene (Hensel et al., Plant Cell 5:553–564, 1993). Therefore, inorder to examine a change in leaf longevity in the ore7 mutant undertreatment with such phytohormones, the progression of senescence wasmeasured by examining chlorophyll contents and photosynthesis activitiesafter the ore7 mutant had been treated with ABA, MeJA and ethylene,respectively. As a result, it was found that, in the wild type, both thechlorophyll content and the photosynthesis activity were significantlyreduced by influencing the phytohormones, resulting in acceleratedsenescence. However, in the ore7 mutant, the effect of the phytohormoneswas highly reduced, so that the ore7 mutant can have an extendedlongevity even if it is treated with the senescence-acceleratinghormones (see FIGS. 3 and 4). Furthermore, results of analysis of SEN4gene, a measure of senescence molecules, indicated that the expressionof SEN4 in the wild type is highly increased, compared to the ore7mutant (see FIG. 5). This suggests that the longevity-extending effectis exhibited at the physiological level and at the molecular level.

In the other embodiment of the present invention, a gene involved in anextension in longevity of plants was isolated from the ore7 mutant by aplasmid rescue method (Weigel et al., Plant Physiology, 122:1003–1014,2000). A nucleotide sequence of a DNA fragment located around a siteinserted with T-DNA that had been isolated by this plasmid resque methodwas then determined.

The determined nucleotide sequence was searched with a genome databaseof Arabidopsis thaliana. As a result, an open reading frame (ORF)located most adjacent to the enhancers was found. It was determined thatthe isolated gene is the gene which has a single exon and consists of936 nucleic acids encoding 311 amino acids. This gene was termed ‘ORE7’.A nucleotide sequence of the ORE7 gene is represented by SEQ ID NO: 1. Adatabase search for a peptide sequence deduced from the determinedsequence of the OER7 gene identified that a protein expressed from theORE7 gene consists of 311 amino acids, as represented by SEQ ID NO: 2,and contains an AT-hook motif. It is known that the AT-hook motif isbound to an AT-rich region of a promoter of a target gene so that itdirectly serves as a transcription factor, or as a transcriptionregulator aiding the transcription factor in getting close to thepromoter, or involves in chromosome architecture like histone (Aravindet al., Nucleic Acids Res. 26(19): 4413–4421, 1998). Furthermore, it wasfound that the ORE7 protein has glycine-, histidine-, glutamine-, andglutamic acid-rich motifs which are commonly found in the transcriptionregulators (Abraham et al., Gene, 255:389–400, 2000; and Fujimoto etal., Biochem. Biophys. Res. Commun., 280(1): 164–171, 2001).

In order to verify whether the senescence-delaying phenotype of the ore7mutant is caused by overexpression of the ORE7 gene, northern blotanalysis was carried out using the ORE7 gene as a probe. As a result, inthe wild type, the expression of the ORE7 gene was not observed, but inthe ore7 mutant, the overexpression of the ORE7 gene was observed (seeFIG. 7). Furthermore, the ORE7 gene was re-introduced into the wild typeArabidopsis thaliana so as to be overexpressed, and examination wascarried out to determine whether the leaf senescence-delaying phenotypeis reproduced. At this time, as s vector, a pCAMBIA3301 vector ispreferably used, although the known vectors for plant transformation mayalso be used without limitation. Moreover, as a host to be transformed,although all microorganisms belonging to an Agrobacterium sp. May beused without limitation, Agrobacterium tumefacience strain was termed‘pAT-ORE7’ and deposited under the accession number KCTC 10032BP on Aug.8, 2001 with the Korean Collection for Type Cultures (KCTC) at theKorean Research Institute of Bioscience and Biotechnology (KRIBB), #52,Oun-dong, Yusong-ku, Taejon 305–333, Republic of Korea.

Then, the wild type Arabidopsis thaliana (Columbia) was transformed withthe pAT-ORE7 by the floral tip method (Clough et al, Plant J., 16(6):735–743, 1998). It was found that the longevity-extending phenotype isreproduced in the transformed Arabidopsis thaliana. This suggests thatthe ORE7 gene according to the present invention is the gene whichinhibits leaf senescence in Arabidopsis thaliana.

Sequencing of a protein expressed from the ORE7 gene of the presentinvention showed that sequence found in the transcription factors ortranscription regulators is present in amino acid sequence of theprotein. Accordingly, since the ORE7 protein expressed from the ORE7gene was deduced to be the transcription factor or transcriptionregulator that regulates the expression of genes in nuclei, the presentinventors confirmed whether the ORE7 protein migrates into the nuclei.For this, a recombinant plasmid which allows the ORE7 gene of thepresent invention to be expressed in the form of a fusion protein with agreen fluorescence protein (GFP) was constructed by a method of Kain etal. (Kain et al. Biotechniques, 19(4): 650–655, 1995). Observation witha fluorescence microscope showed that the expression of GFP occurs inthe nuclei (see FIG. 8). This suggests that the ORE7 protein migratesinto the nuclei and functions therein.

The ORE7 gene of the present invention may function as the transcriptionrepressor for inhibiting the transcription factor which regulates theinitiation of leaf senescence, and also as a negative regulator directlyinhibiting the initiation and progression of senescence. In addition,the ORE7 gene of the present invention may exhibit a function ofincreasing hormones, such as cytokinin or auxin, senescence inhibitoryhormones, thereby indirectly inhibiting senescence.

Meanwhile, the present invention provides a method for extending thelongevity of plants, by transforming the plants with a vector that wasconstructed in such a manner that the ORE7 gene is overexpressed. As amethod for producing the transgenic plants in which the ORE7 gene wasoverexpressed, plant transformation methods known in the art can beused. For example, an Agrobacterium-mediated transformation method usinga binary vector for plant transformation introduced with the ORE7 genecan be used. In addition, when a vector not containing a T-DNA region isused, electroporation, microparticle bombardment, polyethyleneglycol-mediated uptake and the like may be used.

Plants whose longevity can be extended by the method of the presentinvention include food crops comprising rice plant, wheat, barley, corn,bean, potato, Indian bean, oat and Indian millet; vegetable cropscomprising Arabidopsis sp., Chinese cabbage, radish, red pepper,strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, welshonion, onion and carrot; special crops comprising ginseng, tobaccoplant, cotton plant, sesame, sugar cane, sugar beet, Perilla sp., peanutand rape; fruit trees comprising apple tree, pear tree, jujube tree,peach tree, kiwi fruit tree, grape tree, citrus fruit tree, persimmontree, plum tree, apricot tree and banana tree; flower crops comprisingrose, gladiolus, gerbera, carnation, chrysanthemum, lily and tulip; andfodder crops including ryegrass, red clover, orchardgrass, alfalfa,tallfescue and perennial ryegrass, etc. Particularly, when the method ofthe present invention is applied to edible greens or fruits such astomatoes, which have a thin pericarp and thus show rapid deteriorationin quality caused by senescence, and to plants whose leaf is mainlymarketed, it effectively increases the storage efficiency of the plants.

Furthermore, the ORE7 gene and ORE7 protein of the present invention areuseful for investigation of senescence-associated genes or senescenceinhibitory substances in plants. In addition, the gene of the presentinvention can be used to investigate senescence inhibitory substances byinvestigating substances binding to the genes of the present inventionor substances inhibiting or activating the expression of the ORE7 gene.Specifically, investigation can be performed by various conventionalmethods including DNA chip, protein chip, polymerase chain reaction(PCR), northern blot analysis, southern blot analysis, western blotanalysis, enzyme-linked immunosorbent assay (ELISA) and 2-D gel analysisand the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph illustrating leaf senescence depending on time,in the wild type Arabidopsis thaliana (Col) and in thelongevity-extended mutant ore7.

FIG. 1B is a graph showing changes in photosynthesis activity dependingon time, in the wild type Arabidopsis thaliana (Col) and in thelongevity-extended mutant ore7.

FIG. 1C is a graph showing changes in chlorophyll content depending ontime, in the wild type Arabidopsis thaliana (Col) and in thelongevity-extended mutant ore7.

FIG. 1D shows the results of northern blot analysis illustrating theexpression patterns of senescence-associated genes and aphotosynthesis-associated gene depending on time, in the wild typeArabidopsis thaliana (Col) and in the longevity-extended mutant ore7.

SAG12: a senescence-associated gene

SEN4: a senescence-associated gene

Cab: a gene of chlorophyll a/b binding protein

FIG. 2A is a photograph illustrating leaf senescence depending on timeafter dark treatment, in the wild type Arabidopsis thaliana (Col) and inthe longevity-extended mutant ore7.

FIG. 2B is a graph showing a change in photosynthesis activity dependingon time after dark treatment, in the wild type Arabidopsis thaliana(Col) and in the longevity-extended mutant ore7.

FIG. 2C is a graph showing a change in chlorophyll content depending ontime after dark treatment, in the wild type Arabidopsis thaliana (Col)and in the longevity-extended mutant ore7.

FIG. 2D shows the results of northern blot analysis illustrating theexpression pattern of a senescence-associated gene (SEN4) in the wildtype Arabidopsis thaliana (Col) and in the longevity-extended mutantore7 after dark treatment.

0D: before dark treatment

4D: at 4 days after dark treatment

FIG. 3 is a graph showing a change in photosynthesis activity dependingon time, after treatment with a MES buffer solution (negative controlgroup) (A), MeJA (B), ABA (C) and ethylene (D), which aresenescence-accelerating hormones, in the wild type Arabidopsis thaliana(Col) and the longevity-extended mutant ore7.

FIG. 4 is a graph showing a change in chlorophyll content depending ontime, after treatment with MES buffer solution (negative control group)(A), MeJA (B), ABA (C) and ethylene (D), which aresenescence-accelerating phytohormones, in the wild type Arabidopsisthaliana (Col) and the longevity-extended mutant ore7.

FIG. 5 shows the results of northern blot analysis illustrating theexpression pattern of a senescence-associated gene (SEN4) depending ontime, at 0, 3, 4 and 5 days after treatment with MeJA, ABA and ethylene,respectively, which are senescence-accelerating hormones, in the wildtype Arabidopsis thaliana (Col) and the longevity-extended mutant ore7.

C: a control group not treated with the senescence-accelerating hormones

T: a group treated with the senescence-accelerating hormones.

FIG. 6 is a scheme showing that an activation tagging vector pSKI015 isinserted into a genome of the longevity-extended mutant ore7.

E: an enhancer

BAR: a herbicide-resistant gene

pBS: a region containing a replication origin of E. coli and anampicilin-resistant gene

FIG. 7 shows the results of northern blot analysis illustrating theexpression of an ORE7 gene in the wild type Arabidopsis thaliana (Col)and in the longevity-extended mutant ore7, in which 28S is a controlgroup.

FIG. 8 is a photograph showing migration of a GFP-ORE7 fusion proteininto a nucleus, in an epidermal cell of onions, observed under afluorescence-(A and B) and an optical microscope (C and D).

A: a photograph of 35S-GFP (a positive control group)

B: a photograph of 35S-ORE7-GFP

C: a photograph of 35S-GFP (a positive control group)

D: a photograph of 35S-ORE7-GFP

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will hereinafter be described in further detail byexamples. It should however be borne in mind that the present inventionis not limited to or by the examples.

EXAMPLE 1 Selection of Longevity-extended Mutant from Arabidopsisthaliana

First, in order to induce mutations in Arabidopsis thaliana, pSKI015(Weigel et al., Plant Physiology, 122: 1003–1014, 2000; obtained fromWeigel's Lab., USA), an activation tagging vector, was introduced intoan Agrobacterium tumefacience ABI strain (obtained from Amasino's Lab.,USA) by the known electroporation method, and then cultured in a mediumcontaining kanamycin and carbenicillin in order to select transformants.Following this, the wild type Arabidopsis thaliana (Columbia) wastransformed with the Agrobacterium tumefacience ABI strain inserted withpSKI015, according to the floral dip method (Clough et al., Plant J.,16(6): 735–743, 1998). The transformed Arabidopsis thaliana was bred,thereby obtain seeds, and herbicide-resistant transformants wereselected from the seeds. Approximately 5000 T1 lines were then grown ina greenhouse at a controlled temperature of 23° C., and the yellowing ofleaves, caused by a reduction in chlorophyll according to age-dependentplant senescence, was observed with the naked eye. One line of mutantswhich had a slow yellowing rate compared to the wild type was thenselected. This selected mutant was termed ‘ore7’. FIG. 1A shows thesenescence pattern of leaves depending on time, in the wild typeArabidopsis thaliana and in the longevity-extended mutant ore7.

EXAMPLE 2 Examination on Expression of Characters in Longevity-extendedMutant ore7

In order to verify a longevity-extending character of the ore7 mutant, arosette leaf 3 of T2 generation plant was observed with the naked eye,every five days, after 20 DAG, and measured for chlorophyll content,photosynthesis activity. The results were compared with those of thewild type Arabidopsis thaliana. Sample groups in this case were 25independent leaves obtained from each individual.

2-1) Measurement of the Chlorophyll Content

In order to measure the chlorophyll content, the respective sampleleaves were boiled in 95% ethanol at 80° C., thereby extractingchlorophyll. The chlorophyll content was measured at absorbance of 648nm and 664 nm, and expressed as chlorophyll concentration per freshweight of leaves (Vermon et al., Anal. Chem., 32:1142–1150, 1960). Asshown in FIG. 1B, results showed that the chlorophyll content of thewild type was rapidly decreased after 25 DAG and become 0% at 40 DAG,whereas ore7 mutant exhibited a chlorophyll content of more than 70% ofthe initial chlorophyll content even at 40 DAG.

2-2) Measurement of Photosynthesis Activity

In order to measure photosynthesis activity, a method of Oh et al. (OhS. A. et al., Plant Mol. Biol., 30: 939, 1996), was used. First, leavesat the respective DAG were dark-treated for 15 minutes and measured forfluorescence of chlorophylls using Plant Efficiency Analyzer(Hansatech). The photosynthesis activity was expressed as photochemicalefficiency of photosystem II (PS II) using a fluorescent property ofchlorophylls. The photochemical efficiency was expressed as the ratio ofmaximum variable fluorescence (Fv) to maximum value of fluorescence (Fm)(Fv/Fm). As the ratio is increased, the photosynthesis activityimproves. As shown in FIG. 1C, results indicated that, in the wild type,the photosynthesis activity was started to reduce rapidly after 30 DAGs,and almost lost after 40 DAG, whereas in the ore7 mutant, it was startedto reduce after 40 DAG and maintained at about 60% even at 70 DAG due tovery slow reduction patterns thereof.

According to the said results, the ore7 mutant has phenotypes of farlonger leaf longevity than the wild type. This longevity-extendingeffect can be verified from the fact that the biochemical changesaccording to senescence, expressed as a reduction in chlorophyll contentand a reduction in photosynthesis activity, occur later than in the wildtype.

EXAMPLE 3 Examination of Expression of Senescence-associated Genes inore7 Mutant

In order to compare the expression of senescence-associated genes (SAGs)in the ore7 mutant with that in the wild type, the expression patternsof the respective SAG genes depending on the passage of time during aprocess of leaf development were identified by northern blot assay.Total RNA extracted from respective leaves at 22, 26, 30, 34 and 38 DAGusing Tri-reagent (Sigma) was used as samples (Woo H. R. et al., PlantCell, 13:1779–1790, 2001). 10 μg of RNA was loaded in every lane, and aSAG12 gene, a SEN4 gene and a Cab gene were used as probes.

Results showed that the expression of a photosynthesis-associated gene,such as chlorophyll a/b binding protein (Cab) was reduced in proportionto senescence with the passage of time, as shown in FIG. 1D. However, inthe ore7 mutant of the present invention, there were little or nochanges in the expression patterns of these genes. Meanwhile, it wasfound that, in the wild type, the expression of varioussenescence-associated genes, such as SAG12 and SEN4, was increased withthe passage of time, but in the ore7 mutant, the expression patterns ofthe senescence-associated genes were not substantially increased in thesame time. This fact suggests that the ore7 mutant delays the initiationof senescence at the physiological level and also at the molecularlevel, thereby extending the leaf longevity. In addition, the aboveresults were coincident with the results of study indicating thatanabolic activity such as photosynthesis and self-maintenance activityare increased with the growth of leaves and then decreased at thesenescence stage (H. G. Nam, Curr. Opin. Biotech, 8:200, 1997).

EXAMPLE 4 Examination of a Change in Leaf Longevity of ore7 MutantAccording to Dark Treatment

A change in leaf longevity of the ore7 mutant under dark treatment knownas accelerating senescence was examined by measuring changes inphotosynthesis activity and chlorophyll content.

12 independent leaves at 25 DAG were detached from the wild typeArabidopsis thaliana and the ore7 mutant, and floated in 2 ml of 3 mM2-[N-morpholino]-ethanesulfonic acid buffer, pH 5.8 hereinafter,referred to as “MES buffer”). The resulting leaves were measured forphotosynthesis activity and chlorophyll content, every day, in the samemanner as in Example 2, while storing in a light impermeable box at 22°C. Results showed that the photosynthesis activity in the wild type wasdecreased to 60% at 5 days after the dark treatment, but the ore7 mutantmaintained the photosynthesis activity of 97%, as shown in FIG. 2B. Inaddition, it was found that the reduction pattern of the chlorophyllcontent became blunt in the case of the ore7 mutant, similar to thephotosynthesis activity, so that the ore7 mutant exhibited thechlorophyll content of 75% that is three times as large as that of thewild type whereas the wild type exhibited the chlorophyll content of25%, at 5 days after the dark treatment, as shown in FIG. 2C.

Moreover, the expression of a SEN4 gene, a measure of senescencemolecules, was examined in the same manner as described in Example 3.Results indicated that the expression of SEN4 in the wild type washighly increased, compared to the ore7 mutant, as shown in FIG. 2D.

EXAMPLE 5 Changes in Leaf Longevity of ore7 Mutant According toTreatment with Phytohormones

Further, in the present invention, a change in leaf longevity of theore7 mutant according to treatment with phytohormones, such as ABA, MeJAand ethylene that are involved in senescence regulation was examined bymeasuring changes in photosynthesis activity and chlorophyll content. 12independent leaves at 25 DAG were floated in 2 ml of MES buffercontaining 50 μM ABA or 50 μM MeJA. Treatment with ethylene was carriedout by culturing Arabidopsis thaliana in a glass box containing 4.5 μMethylene gas. As a negative control group, MES buffer not containing ABAor MeJA was used. The treatments with phytohormones as described abovewere carried out for three days at 22° C. with continuous exposure tolight. The chlorophyll content and the photosynthesis activity weremeasured in the same manner as described in Example 2.

Results showed that, in the case of the negative control group (treatedwith only MES buffer not containing ABA or MeJA), as shown in FIGS. 3Aand 4A, there are no changes in photosynthesis activities andchlorophyll contents of the wild type and the ore7 mutant. On the otherhand, as shown in FIGS. 3B to 3D, the photosynthesis activity in thewild type was rapidly decreased from 2 days after treatment with ABA,MeJA and ethylene, respectively, but the ore7 mutant exhibited a slightdecrease in the photosynthesis activity. Furthermore, as shown in FIGS.4B to 4D, a reduction in the chlorophyll content became blunt in thecase of the ore7 mutant, similar to the photosynthesis activity. Theseresults suggest that the ore7 mutant has low susceptibility to thesenescence-accelerating hormones.

EXAMPLE 6 Cloning and Sequencing of ORE7 Gene

In order to investigate a gene around T-DNA activated by an enhancer,the genes was isolated from the ore7 mutant according to the plasmidrescue method (Weigel et al., Plant Physiology, 122:1003–1014, 2000).FIG. 6 is a scheme showing that an activation tagging vector pSKI015 isinserted into a genome of the longevity-extended mutant ore7. First, thetotal genomic DNA of the ore7 mutant was isolated by the methoddescribed by Dellaporta et al. (Dellaporta et al., Plant Mol. Biol.Rep., 1:19–21, 1983). After this, 5 μg of genomic DNA was digested withEcoRI restriction enzyme, purified by ethanol precipitation, and thendried. The digested DNA was self-ligated, and transformed into DH5α. Anampicillin-resistant colony was selected out of transformants. A plasmidwas isolated from the selected colony, and a nucleotide sequence of aregion which is 4.0 kb apart from the right border of T-DNA wasdetermined using an oligomer constructed based on a nucleotide sequencedownstream of the EcoRI region which had been used in the above plasmidrescue method. The determined nucleotide sequence was searched withreference to the Arabidopsis thaliana genome database, therebyinvestigating a open reading frame closest to an enhancer. Theidentified open reading frame was termed ‘ORE7 gene’ whose nucleotidesequence is represented by SEQ ID NO: 1. It could be found that proteinexpressed from the ORE7 gene consists of 311 amino acids, as shown inSEQ ID NO: 2. Further, it could be found that an AT-hook motif ispresent at 83–94 region of the amino acid sequence represented by SEQ IDNO: 2, and glycine-, histidine-, glutamine-, and glutamic acid-richmotifs are present at 38–52 and 245–261 regions of the amino acidsequence.

Thereafter, northern blot analysis was carried out using the ORE7 geneas a probe. Total RNAs were extracted from the wild type and the ore7mutant, respectively, using Tri-reagent (Sigma), according to the methodby Woo et al. (Woo H. R. et al. Plant Cell, 13:1779–1790, 2001). 10 μgof RNA was loaded onto a 1.2% agarose/formaldehyde gel, every lane,isolated, then blotted to a nylon membrane. The resulting RNA sample waswashed with 3×SSC for 5 minutes to remove an excess of agarose. Then, inorder to immobilize the RNA sample to the nylon membrane, irradiationwith ultraviolet ray (254 nm, 0.18 J/cm²) was carried out. The resultingblot was subjected to prehybridization and hybridization according tothe method by Park et al. (Park et al., Plant Mol. Biol, 26:1725–1735,1994).

Results showed that there is little or no expression of the ORE7 gene inthe wild type, whereas in the ore7 mutant, the ORE7 gene is expressed atsignificantly high transcription concentration, as shown in FIG. 7.

EXAMPLE 7 Introduction of ORE7 Gene into Wild Type Arabidopsis thaliana

In order to finally determine whether the senescence-delaying phenotypeoccurring in the ore7 mutant is caused by activation of the OER7 gene,experiments in which a DNA fragment of 5.4 kb size containing anenhancer and the ORE7 gene is introduced into the wild type Arabidopsisthaliana were carried out.

For this, the plasmid obtained by the plasmid rescue method was digestedwith BamHI and EcoRI, thereby isolating a DNA fragment containing theORE7 gene and an enhancer. The digested DNA fragment was inserted into apCAMBIA3301 vector (MRC, USA) digested with BamHI and EcoRI. Theresulting vector was termed ‘pORE7/3301’. An Agrobacterium tumefacienceAGL1 strain (Lazo G. R., et al., Biotechnology, 9:963–967, 1991) (ATCCBAA-101) was then transformed with the recombinant vector pORE7/3301.The transformed Agrobacterium tumefacience strain was termed ‘pAT-ORE7’and deposited under the accession number KCTC 10032BP on Aug. 8, 2001with the Korean Collection for Type Cultures (KCTC). Then, the wild typeArabidopsis thaliana (Columbia) was transformed with pAT-ORE7 by thefloral tip method (Clough et al., Plant J., 16(6): 735–743, 1998). Thetransformed Arabidopsis thaliana was bred, thereby obtaining seeds, andherbicide-resistant transformants were selected from the seeds. Thetransformed Arabidopsis thaliana was grown in a greenhouse, and examinedfor senescence-delaying patterns. Results showed that thesenescence-delaying phenotype that was exhibited in the ore7 mutant isreproduced as it is. This suggests that the senescence-delayingphenotype is exhibited by activation of the ORE7 gene, and activation ofthe ORE7 gene in plants can induce senescence delay in the plants.

EXAMPLE 8 Examination on Expression of GFP-ORE7 Fusion Protein inEpidermal Cells of Onions

A polypeptide sequence deduced from the nucleotide sequence of ORE7 genedetermined in Example 6 was searched with databases. Results showed thatit has an AT-hook motif and also glycine-, histidine-, glutamine-, andglutamic acid-rich motifs. These motifs are those found in thetranscription factor or transcription regulator of regulating theexpression of other genes in nuclei. Therefore, the present inventorshave determined whether the ORE7 protein of the present inventionmigrates into the nuclei or not.

First, a gene encoding the total ORE7 protein was amplified by PCR withprimers represented by SEQ ID NO: 3 and SEQ ID NO: 4. The PCR reactionwas initiated by heating to 95° C. for 2 minutes, then subjected to 35cycles under the condition as the following: 94° C. for 30 seconds, 55°C. for 30 seconds, and 72° C. for 1 minute, and completed by finallyamplifying for 10 minute at 72° C. A DNA fragment of a 0.95 kb sizeobtained by the above PCR was isolated by agarose gel electrophoresis,and then was inserted into SmaI and HindIII restriction enzyme sites ofa 326 GFP-3G plasmid (obtained from Professor In-Hwan, Hwang, PohangUniversity of Science and Technology Foundation, Korea) containing agene of a green fluorescence protein (GFP), thereby constructing aplasmid pGFP-ORE7. Following this, the expression of a GFP-ORE7 fusionprotein in epidermal cells of onions was examined according to themethod by Varagona et al (Varagona et al., Plant Cell, 4:1213–1227,1992). For coating of tungsten particles with DNA, a plasmid expressingthe ORE7-GFP fusion protein was purified using a Qiagen column, and then2 μg of DNA was precipitated with tungsten particles in a solutioncontaining 50 μl of 2.5 M CaCl₂ and 20 μl of 0.1 M spermidine. Theprecipitates were washed with 70% ethanol, and then resuspended in 36 μlof 100% ethanol. Subsequently, epidermal cells of onions were placed ona ½ B5 medium in a petri dish in such a manner that the inner coat ofthe onion faced upward. Then, the M-25 tungsten particle coated with theplasmid DNA was subjected to particle bombardment using 1350 p.s.i.(Biorad). The petri dish was wrapped with parafilm, and then incubatedin an incubator at 22° C. for 18 hours. After 18 hours, the expressionof GFP was observed with a fluorescence microscope.

Results showed that the expression of ORE7-GFP occurs within the nuclei,as shown in FIG. 8. This suggests that the ORE7 protein migrates intothe nuclei and functions therein.

INDUSTRIAL APPLICABILITY

As apparent from the foregoing, it was found that thesenescence-delaying phenotype of the ore7 mutant occurs by activation ofthe ORE7 gene, and activation of the ORE7 gene can induce the delay ofsenescence in plants. Plants can be transformed with the ORE7 generegulating the leaf longevity of plants according to the presentinvention, so that the longevity of the plants is increased, therebyimproving productivity and storage efficiency of the plants.Furthermore, the ORE7 gene and the ORE7 protein expressed therefromaccording to the present invention are useful for studies of senescencemechanisms, and for investigation of senescence-associated genes orsenescence inhibitory substances, in plants.

1. An Agrobacterium tumefacience pAT-ORE7 (accession number: KCTC10032BP) transformed with a recombinant vector comprising apolynucleotide comprising a sequence encoding an amino acid sequencerepresented by SEQ ID NO:
 2. 2. A method for increasing the longevity ofleaves of a plant, comprising introducing a polynucleotide comprising asequence encoding an amino acid sequence represented by SEQ ID NO: 2into said plant so that it is overexpressed, and selecting fortransformed plants wherein the longevity of leaves is increased comparedto non-transformed plants of the same species.
 3. The method accordingto claim 2, wherein the polynucleotide comprises the nucleotide sequencerepresented by SEQ ID NO:
 1. 4. The method according to claim 2, whereinthe plants are selected from among food crops, fruit trees, flowercrops, and fodder crops.
 5. The method according to claim 4, wherein theplants are food crops selected from among rice plant, wheat, barley,corn, bean, potato, Indian bean, oat and Indian millet; vegetable cropscomprising Arabidopsis sp., Chinese cabbage, radish, red pepper,strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, welshonion, onion and carrot.
 6. The method according to claim 4, wherein theplants are fruit trees selected from among apple tree, pear tree, jujubetree, peach tree, kiwi fruit tree, grape tree, citrus fruit tree,persimmon tree, plum tree, apricot tree and banana tree.
 7. The methodaccording to claim 4, wherein the plants are flower crops selected fromamong rose, gladiolus, gerbera, carnation, chrysanthemum, lily andtulip.
 8. The method according to claim 4, wherein the plants are foddercrops selected from among ryegrass, red clover, orchardgrass, alfalfa,tallfescue and perennial ryegrass.
 9. The method according to claim 2,wherein the plants are selected from among ginseng, tobacco plant,cotton plant, sesame, sugar cane, sugar beet, Perilla sp., peanut andrape.